Western Blotting Protocol

Matthew Allan

Western Blotting Protocol

I. Prepare protein samplesII. SDS PAGEIII. Membrane transferIV. Preliminary StainingV. Cutting membraneVI. BlockingVII. Primary antibodyVIII.Secondary antibodyIX. Chemiluminescent treatmentX. Imaging

I. SDS PAGE

1. Make a polyacrylamide gel2. Load protein samples into a gel3. Run gel

Make a polyacrylamide gel

1. Determine gel percent (see chart) 8% to 14% best

2. Clean cassette and ensure sealing3. Make corresponding resolving gel

and stacking gel (the latter without TEMED); add resolving gel to cassette up to the notch

4. Wait 30 minutes or until solidified5. Add TEMED to stacking gel and

add on top of resolving gel6. Wait 30 minutes or until solidified7. Remove from cassette; clean up

Load protein samples into a gel

1. Place gel into electrophoresis apparatus; its notch aligns with the notch on the apparatus (short plate faces inwards, spacer plate outwards)

2. Ensure the rear plastic piece fits snugly into apparatus3. Tighten clamps and ensure both sides of internal chamber are sealed4. Place apparatus in plastic container5. Fill middle chamber with running buffer; spill over into larger chamber

until lower electrode is covered6. Pipette loading dye into protein samples (use 1:10 loading dye:protein)

the loading dye contains 10x SDS and beta mercaptoethanol7. Pipette protein samples into corresponding wells, rinsing the tip

between each sample by pipetting the buffer through the tip; add a ladder to the penultimate well and loading dye only to the first and last; all wells should be equivolumetric

Run gel

1. Place the top on the electrophoresis apparatus2. Set the machine for 220 V and run for 40 min3. Periodically check if machine is still running: “ER”

means something is wrong—likely a loose electrical connection

4. Stop it as soon as the loading dye emerges at the bottom of the gel, and don’t let it run any further

II. Membrane transfer

1. Set up membrane and gel stack2. Run electric current3. Discard all materials except the membrane

Set up membrane and gel stack

1. Clean machine with dd water2. For four pieces of filter paper, soak them all at once in

transferring buffer (20% methanol, 10% 10x transferring buffer) and stack them up; do not touch the middle and use a flat comb to press out trapped air bubbles

3. Soak and add the membrane4. Trim off the stacking gel, add the resolving the gel, and

smooth it out5. Add four more filter papers as described above6. Close the machine and place heavy books on top

Run electric current

1. Set the current to 90 mA per gel2. Let the machine run for 1 hour

Discard all materials except the membrane1. The filter paper and gel go into the trash2. NEVER touch the center of the membrane with fingers

or gloves; place it into a plastic bin for staining

III. Preliminary staining

The first time you run the gel, you stain the gel itself with Commassie Blue to equalize the amount of protein loaded into each well. Stain with Ponceau S for membranes for western blotting to calibrate the amount of protein to add to each well.

III. Preliminary staining

Stain with either Ponceau S for membranes for western blotting to calibrate the amount of protein to add to each well

Ponceau S Stain

1. Pour Ponceau S over the membrane in the plastic bin and slosh it around for several seconds, until bands appear

2. Pour out the Ponceau S and wash briefly with ddH2O, but not so much as to remove the Ponceau S

3. Scan the films in to record how much protein is in each lane with the scanner

4. If needed, the membrane can be placed in a protective sleeve of plastic that is cut to size and then stored at 4 degrees

Coomassie Blue Stain

1. Wash the gel with a fixing agent for coomassie blue on the rocking platform for 10 min

2. Pour out the fixing agent and add the commassie blue carefully (it is toxic), then incubate on the rocking platform for 10 min

3. Pour out the commassie blue into the commassie blue waste containers (it cannot go down the drain

4. Add the destain solution and a VWR light dust paper and place on the rocking platform for a variable time, up to overnight, then pour it out

5. Commassie blue gels are not blotted; rather, they determine whether more or less protein should be added to subsequent gels in order to equalize the protein between lanes

IV. Cutting membrane

1. Place membrane in protective sleeve2. Label regions of interest3. Cut apart regions of membrane

Place membrane in protective sleeve1. Cut a piece of the protective plastic sleeve using the

paper trimmer and place the membrane inside the sleeve

Label regions of interest

1. Identify the areas of the membrane in which the proteins of interest will be, using the protein ladder as a guide

2. Outline them with a pen, writing through the plastic sleeve, and label each one with the protein of interest and the lane numbers

Cut apart regions of membrane

1. With the membrane in its sleeve, cut apart the outlined regions with paper trimmer

2. Discard the needless regions of the membrane

V. Blocking

1. Wash membrane2. Incubate with milk solution on rocking platform

Membrane

Milk protein

Protein of interest

Wash membrane

1. Remove each membrane from its plastic sleeve with forceps

2. Place it in a a plastic bin and cover with TBST3. Rock the membrane back and for 5 mins, until the

Ponceau S disappears4. Carefully pour the TBST into the sink

Incubate with milk solution on rocking platform1. Cover the membranes with 5% milk solution in TBST2. Rock on setting 2 for 5 to 10 minutes3. Take the membranes out of the plastic bins

V. Primary antibody

1. Prepare bag with milk and primary antibody2. Seal membrane into bag3. Incubate in vertical rotator

Heavy variableLight variable

Light constantHeavy constant

Specific bindingUnspecific binding

Prepare the bag with milk and primary antibody1. Calculate the correct amount of each primary antibody

to add; typical concentrations range from 1:20000 to 1:500

2. Cut out a section of bag film about 5 cm by 10 cm and seal three sides closed

3. Label each bag with the protein to be blotted for4. Pipette 3 to 4 mL of 5% milk into each bag5. Go to the antibody freezer with a 2 – 20 microliter

pipette and tips6. In the freezer, pipette each primary antibody into its

corresponding bag, or take the antibody to the lab bench and add it there

Seal membrane into bag

1. Place each membrane in its corresponding bag with forceps

2. Press out the bubbles from each bag3. Seal the bag closed, leaving enough space for the

membrane to move around; press on the bag to ensure there are no holes

Incubate in vertical rotator

1. Tape each bag to the vertical rotator in the refrigerator2. Start the rotator, and do not adjust the speed3. Lightly tap each bag to make sure the membranes can

float freely

VI. Secondary antibody

1. Wash membrane in TBST2. Prepare bag with milk and HRP-fused secondary

antibody3. Seal membrane into bag4. Incubate in vertical rotator

Wash membrane in TBST

1. Cover the membranes with TBST2. Rock on setting 2 for 10 minutes or setting 3 for 5

minutes3. Carefully pour the TBST into the sink4. Repeat steps 1 to 3 twice more

Prepare the bag with milk and secondary antibody1. Calculate the correct amount of each secondary antibody to

add; typical concentrations range from 1:2000 to 1:5002. Cut out a section of bag film about 5 cm by 10 cm and seal

three sides closed3. Label each bag with the protein to be blotted for4. Pipette 3 to 4 mL of 5% milk into each bag5. Go to the antibody freezer with a 2 – 20 microliter pipette

and tips6. In the freezer, pipette each primary antibody into its

corresponding bag; every primary antibody comes from an animal, and every species has a unique heavy constant region; the secondary antibody must match the species that made first antibody for it to bind

Seal membrane into bag

1. Place each membrane in its corresponding bag with forceps

2. Press out the bubbles from each bag3. Seal the bag closed, leaving enough space for the

membrane to move around; press on the bag to ensure there are no holes

Incubate in vertical rotator

1. Tape each bag to the vertical rotator in the refrigerator2. Start the rotator, and do not adjust the speed3. Lightly tap each bag to make sure the membranes can

float freely

VI. Chemiluminescent treatment

1. Combine the two reagents2. Add reagent mixture to membranes3. Align membranes on protective sleeve

The HRP, part of the secondary antibody, causes the luminol to glow

Combine luminol and substrate for Western Blot1. Take out the box of the two reagent bottles from the

refrigerator2. Into a microcentrifuge tube, pipette 100 microliters

per membrane of each reagent3. Put the reagents back in the refrigerator

Add the reagent mixture to the membranes1. Place the membranes on a piece of Saran Wrap;

ensure that the text is readable and the membranes are protein-face-up

2. Pipette-drip the reagent mixture onto each membrane, 200 microliters per membrane, covering evenly and completely

3. Incubate for 1 minute

Align membranes on protective sleeve

1. Move the membranes to an open piece of protective sleeve with forceps

2. Position them such that they are as close as possible without overlapping and right beside the guide strip affixed to the paper

3. Close the sleeve and tape it shut

VI. Imaging

1. Take membranes and film to the darkroom2. Quickly press film against membrane3. Repeat as necessary4. Feed film through developing machine

Take membranes and film to the darkroom1. Take the film out of the drawer to the left of the SDS

PAGE machines2. Walk to the darkroom in South Frear, flip on the

normal light, make sure the exposure bench is clear and the exposure machine is set to “Day,” open up the black sleeve with the membranes, and then switch to the dark light

3. Wait for the eyes to adjust

Quickly press the film against the membrane1. Open the film box and remove one piece of film2. Holding the black paper sleeve open, quickly place the

film in position on top of the membranes’ protective plastic sleeve

3. Keeping one hand on the film at all times to prevent it from moving, place the right hand on the film, remove the left hand from the film, and then, with the left hand, press the black sleeve top flap down onto the film; finally, remove the right hand to allow the sleeve to cover the film entirely

4. Hold for the predetermined amount of time

Repeat as necessary

1. Different regions of the same piece of film can be used for two or more images

2. Different membranes can be imaged3. Different exposure times, from 2 seconds to 10

minutes, can be used

Feed film through developing machine

1. Once all images have been collected, place each piece of film, one at a time, on the right side feed-in tray

2. The film will automatically be pulled in and developed3. Once the machine beeps, the next piece of film can be

fed in4. After the final beep, the film has been fully developed

and is no longer light-sensitive5. Ensure that the box of film is closed correctly before

turning on the normal light6. Remove the film from the developing machine7. Analyze the results

References

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6376.pdf